Human pluripotent stem cells (hPSCs), which include embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), offer unprecedented promise for cardiac repair; however, many hurdles must be overcome before clinical application is possible. Current methods for differentiating cardiomyocytes (CMs) from hPSCs result in heterogeneous populations of cells, including undifferentiated or non-CM cells, which may pose a risk for tumor or aberrant tissue formation. For therapeutic use, ventricular CMs (vCMs) are ideal because vCMs are the most extensively affected cell type in myocardial infarction and the major source for generating cardiac contractile forces. Although various strategies have been attempted for CM enrichment, these strategies have drawbacks in terms of purity or specificity. We recently developed a novel technology to purify CMs by targeting CM-specific intracellular mRNAs using molecular beacons (MBs), which will enable isolation of the desired CMs for use in therapy. Another obstacle to cell therapy is low retention of transplanted cells in injured myocardium. To overcome this problem, engineering of CMs with biomaterials or co-transplantation with other cells has been attempted. My co-investigator and I have been working to develop technologies for engineering cardiovascular cells with peptide amphiphile (PA)-based nanostructured biomaterials for enhancing the effects of cell therapy. The goal of this proposal is to develop a strategy for cardiac repair with hPSC-derived vCMs by two novel approaches: to purify hPSC-derived vCMs with MBs and to engineer these CMs with self-assembled nanomatrix gels and other cells including hPSC-derived ECs and fibroblasts. In aim 1, we seek to purify vCMs from differentiating hPSCs using specific MBs. In aim 2, we will engineer purified hPSC-derived CMs, hPSC-derived ECs and other supportive cells with extracellular matrix-mimicking self-assembled nanomatrix gel which contains growth factors to enhance cell viability, vascularization, and cell maturation, and determine the optimal cell- nanomatrix constructs. In aim 3, we will determine the therapeutic effects of engineered hPSC- derived cardiovascular cell constructs on injured myocardium and elucidate the underlying mechanisms. We anticipate that the results of the proposed experiments will yield new insight into the role of novel stem cell therapy for cardiac repair.